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Water is indispensable for life, yet many lack access to clean drinking water, resulting in fatalities from waterborne bacterial infections. Precise assessment of microbial abundance and viability in natural aquatic environments is vital. Adenosine triphosphate (ATP) serves as a parameter for viability assessments due to its presence in viable bacterial cells as an energy carrier. Traditional ATP detection methods involve chemical or enzymatic extraction, followed by measurement of light emission via the Luciferin–Luciferase complex. However, these methods are costly, present a low stability, require specialized equipment, and entail complex sample pretreatment. To overcome these limitations, we developed a biosensor based on aptamers, nucleic acid sequences with specific target-molecule-binding capabilities. Aptamers offer advantages such as an enhanced stability, a lower cost, and ease of design compared to antibodies. Recently, ATP has been used for aptamer selection testing. Our proposed biosensor utilizes a structure-switching ATP-binding DNA nanoswitch with two functional domains: a catalytic DNA-zyme domain and an ATP-binding aptamer domain. In the presence of ATP, its binding to the aptamer domain triggers the activation of the DNA-zyme domain, which is exploited for chemiluminescence (CL) detection. Integrating functional DNA biosensors with microfluidic paper-based analytical devices (µPADs) holds promise for point-of-care (POC) applications. However, achieving proper DNA binding on paper remains challenging, often requiring solution-based assay protocols, leaving µPADs for final signal readout. Here, we introduce an origami µPAD with preloaded dried reagents, allowing for on-paper assay execution upon sample addition and proper folding. Paper functionalization strategies and assay protocols were optimized to ensure simple and straightforward detection of ATP, employing a portable charge-coupled device (CCD) camera for CL detection. Calibration curves plotted against the logarithm of ATP concentration in the range of 1 to 500 µM facilitated determination of the assay’s limit of detection (LOD), which was found to be 3 µM.

Abstract Background The prevalence and death rate arising from malaria infection, and emergence of other diseases showing similar symptoms to malaria require the development of malaria-specific and sensitive devices for its diagnosis. To address this, the design and fabrication of low-cost, rapid, paper-based analytical devices (µPAD) using surface-immobilized aptamers to detect the presence of a recombinant malarial biomarker—Plasmodium falciparum lactate dehydrogenase (rPfLDH)—is reported in this study. Methods Test zones on paper surfaces were created by covalently immobilizing streptavidin to the paper, subsequently attaching biotinylated aptamers to streptavidin. Aptamers selectively bound rPfLDH. The measurement of captured rPfLDH enzyme activity served as the means of detecting this biomarker. Enzyme activity across three replicate sensors was digitally quantified using the colorimetric Malstat assay. Results Screening of several different aptamers reported in the literature showed that aptamers rLDH7 and 2008s immobilized in this manner specifically recognised and captured PfLDH. Using rLDH7, the sensitivity of the µPAD sensor was evaluated and the µPAD sensor was applied for preferential detection of rPfLDH, both in buffered solutions of the protein and in spiked serum and red blood cell lysate samples. In buffered solutions, the test zone of the µPAD sensor exhibited a K D of 24 ± 11 nM and an empirical limit of detection of 17 nM, respectively, a limit similar to commercial antibody-based sensors exposed to rPfLDH. The specific recognition of 133 nM rPfLDH in undiluted serum and blood samples was demonstrated by the µPAD. Conclusion The reported µPAD demonstrates the potential of integrating aptamers into paper-based malarial rapid diagnostic tests. Graphical Abstract The assembly of µPAD sensors using APTEC assay principles for the detection the malarial biomarker, lactate dehydrogenase enzymes from Plasmodium falciparum (PfLDH). The aptamers immobilized at the test zones capture the PfLDH in samples. After washing the unbound sample components from the zones, Malstat assay reagents are added for colour development, proportional to the amount of captured PfLDH.

Water is indispensable for life, yet many lack access to clean drinking water, resulting in fatalities from waterborne bacterial infections. Precise assessment of microbial abundance and viability in natural aquatic environments is vital. Adenosine triphosphate (ATP) serves as a parameter for viability assessments due to its presence in viable bacterial cells as an energy carrier. Traditional ATP detection methods involve chemical or enzymatic extraction, followed by measurement of light emission via the Luciferin–Luciferase complex. However, these methods are costly, present a low stability, require specialized equipment, and entail complex sample pretreatment. To overcome these limitations, we developed a biosensor based on aptamers, nucleic acid sequences with specific target-molecule-binding capabilities. Aptamers offer advantages such as an enhanced stability, a lower cost, and ease of design compared to antibodies. Recently, ATP has been used for aptamer selection testing. Our proposed biosensor utilizes a structure-switching ATP-binding DNA nanoswitch with two functional domains: a catalytic DNA-zyme domain and an ATP-binding aptamer domain. In the presence of ATP, its binding to the aptamer domain triggers the activation of the DNA-zyme domain, which is exploited for chemiluminescence (CL) detection. Integrating functional DNA biosensors with microfluidic paper-based analytical devices (µPADs) holds promise for point-of-care (POC) applications. However, achieving proper DNA binding on paper remains challenging, often requiring solution-based assay protocols, leaving µPADs for final signal readout. Here, we introduce an origami µPAD with preloaded dried reagents, allowing for on-paper assay execution upon sample addition and proper folding. Paper functionalization strategies and assay protocols were optimized to ensure simple and straightforward detection of ATP, employing a portable charge-coupled device (CCD) camera for CL detection. Calibration curves plotted against the logarithm of ATP concentration in the range of 1 to 500 µM facilitated determination of the assay's limit of detection (LOD), which was found to be 3 µM. [ABSTRACT FROM AUTHOR]

Background: The prevalence and death rate arising from malaria infection, and emergence of other diseases showing similar symptoms to malaria require the development of malaria-specific and sensitive devices for its diagnosis. To address this, the design and fabrication of low-cost, rapid, paper-based analytical devices (µPAD) using surface-immobilized aptamers to detect the presence of a recombinant malarial biomarker—Plasmodium falciparum lactate dehydrogenase (rPfLDH)—is reported in this study. Methods: Test zones on paper surfaces were created by covalently immobilizing streptavidin to the paper, subsequently attaching biotinylated aptamers to streptavidin. Aptamers selectively bound rPfLDH. The measurement of captured rPfLDH enzyme activity served as the means of detecting this biomarker. Enzyme activity across three replicate sensors was digitally quantified using the colorimetric Malstat assay. Results: Screening of several different aptamers reported in the literature showed that aptamers rLDH7 and 2008s immobilized in this manner specifically recognised and captured PfLDH. Using rLDH7, the sensitivity of the µPAD sensor was evaluated and the µPAD sensor was applied for preferential detection of rPfLDH, both in buffered solutions of the protein and in spiked serum and red blood cell lysate samples. In buffered solutions, the test zone of the µPAD sensor exhibited a KD of 24 ± 11 nM and an empirical limit of detection of 17 nM, respectively, a limit similar to commercial antibody-based sensors exposed to rPfLDH. The specific recognition of 133 nM rPfLDH in undiluted serum and blood samples was demonstrated by the µPAD. Conclusion: The reported µPAD demonstrates the potential of integrating aptamers into paper-based malarial rapid diagnostic tests. [ABSTRACT FROM AUTHOR]

Highlights • We set up an affordable method for the production inkjet printed conductive lines. • We tested plastic and paper as flexible sensors substrates using a consumer printer. • We characterized the printed lines morphology and their conductivity over time. • We defined an easy and rapid functionalization protocol for IDE printed devices. • Finally, we detected Ampicillin antibiotic in real milk samples by EIS measurements. Abstract Inkjet printing is an established technology that has been revisited in recent years for the low-cost and easy production of electrical sensing and biosensing systems. With this technique, it is possible to produce flexible microsystems on eco-compatible substrates such as polymers and paper. In this sense, we investigated the printing performances of a common office inkjet printer and of a commercial conducting ink, to produce aptamer-based electrochemical biosensors for antibiotics detection. During our analysis we characterized both the morphological and electrical properties of the printed electrodes, considering also their stability over time and tuning all the parameters to obtain reproducible micrometric systems. The produced devices proved to be able to undergo aptamer functionalization and to detect Ampicillin, a common used antibiotic, in few minutes with a LOD of 10 μg/ml in milk. Therefore, in this preliminary work we demonstrated the feasibility of low-cost customized inkjet-printed biosensors, and their possible use to prevent both antibiotic contaminations and antibiotic resistance development. [ABSTRACT FROM AUTHOR]